Influenza A viruses (lAV) and Ebola viruses (EBOV) are negafive-sense RNA viruses that cause human infecfions associated with respiratory disease or hemorrhagic fever, respectively. Both viruses have small genomes, encoding only 11-12 (lAV) or 8 (EBOV) known viral proteins; however, the idenfification of novel proteins in recent years suggests that these viruses may encode additional uncharacterized genes that are transcribed or transcribed/translated, via non-conventional strategies. We hypothesize that these genes and their products likely contribute to the viral life cycle. Therefore, the goal of research project 2 (RP 2) is to identify and determine the functions of uncharacterized lAV and EBOV genes. In Aini 1, we will experimentally prove that hypothetical genes can be expressed from the genomes of lAV and EBOV at the protein level. For all uncharacterized gene categories; 1) proven hypothefical gene candidates, 2) unknown open reading frame candidates (i.e., viral proteins already known to be expressed but not yet functionally characterized), and 3) noncoding viral RNA candidates, we will assess the biochemical function of these uncharacterized gene candidates, including using apoptosis assays in our laboratory. Additional biochemical assays will be perfonned in RP 1 - Baric to examine the role of uncharacterized genes in interferon and tolllike receptor signaling and in RP 3 - Damania to examine the role of uncharacterized genes in inflammasome and p53 signaling. Collecfively, all three RPs will contribute various assays to determine the biochemical funcfion of the uncharacterized genes in this proposal. By using reverse genefics techniques developed in our laboratory for lAV and EBOV, we will generate mutant viruses that no longer encode the uncharacterized gene product. We will then compare the growth kinetics and host responses to infection of wild-type virus and mutant viruses in cell culture (Aim 2) and in a mouse model (Aim 3). In addition, we will perform confocal microscopy imagining (Aim 2) to determine the subcellular localization of the potentially novel lAV and EBOV gene products, and also of potentially novel SARS coronavirus (RP 1) and human herpesviruses-8 (RP 3) gene products. Such microscopy studies will aid in determining the function of these